Real-time optical system for polymerase chain reaction

ABSTRACT

An improved device and system for facilitating polymerase chain reaction including a light source, detector, waveguide, and filters that occupy minimal space and facilitate reduced sample read time and rapid reading of multiple light wavelengths.

FIELD OF THE INVENTION

The present invention relates generally to devices and systems forfacilitating polymerase chain reactions.

BACKGROUND OF THE INVENTION

A number of optical detection systems have been developed for use inqualitative and quantitative nucleic acid measurements. Many suchsystems involve the use of fluorescing agents (fluorescent probes,markers, labels, or dyes) in which the resulting signal intensities aregenerally proportional to the reaction products of polymerase chainreaction (PCR) amplification.

As an example, U.S. Pat. No. 5,928,907 describes a system forfacilitating real-time fluorescence-based measurements of nucleic acidamplification products utilizing a lens co-axially disposed with a fiberoptic cable for focusing a single color excitation beam into the volumeof a sample. U.S. Pat. No. 6,144,448 describes a fluorescence detectingdevice including direct fiber optic connections between a single lightsource, container holder and single fluorescence detector.

U.S. Pat. No. 7,315,376 describes a sample holder provided together withan optical manifold having an excitation source, a photo receiver, orboth, for each sample. U.S. Pat. No. 7,507,575 describes a dataacquisition device and a detection device coupled to the dataacquisition device. The detection device includes a plurality ofremovable optical modules and a rotating disk having a plurality ofprocess chambers having a plurality of species that emit fluorescentlight at different wavelengths. U.S. Pat. No. 8,137,616 describes asystem for performing multi-color real time PCR, comprising a flexiblereal time PCR instrument and a specific composition or reaction forperforming multiplex PCR.

There remains a need for an improved system and device for facilitatingpolymerase chain reaction that allows for detection of stationarysamples, reduced sample read time and optionally simultaneous reading ofmultiple light wavelengths, resulting in an increase in the speed withwhich amplification and quantification take place. There is a furtherneed for instruments that include multiple light sources and detectorsthat occupy minimal space and require little or no ancillaryinstrumentation for facilitating light provision fluorescence detection,or movement of samples to read different samples or fluorescentwavelengths. There is a need for instruments that do not rely on preciseand/or complex alignments of reflective components such as mirrors,enclosures, beam splitters, dichrotic/dichroric filters, ormicroelectronic mirrors for light routing. There is also a need forinstruments that facilitate PCR and detection without direct connectionbetween a sample holder and fiber optic cable.

SUMMARY OF THE INVENTION

The present teachings meet one or more of the above needs by providingan instrument for performing polymerase chain reaction with real-timedetection, including a light source, detector, waveguide, a filters thatoccupy minimal space and facilitate detection of stationary samples,reduced sample read time, and simultaneous reading of multiple lightwavelengths.

The present teachings provide for a device comprising a polymerase chainreaction instrument that includes a sample holder configured to receiveone or more sample tubes that each have at least one portion that isgenerally optically transparent, and that receives a biological samplehaving a nucleic acid to be amplified and at least one fluorescing agentthat interacts with the nucleic acid during amplification and that emitslight upon excitation by light of a known wavelength. The instrument mayfurther include at least one light emitting diode device (device beingan integrated assembly of light emitting diodes or a compact group oflight emitting diodes) that is carried on at least one supportsubstrate, is in electrical communication with a power source, and isadapted to emit light at a plurality of different wavelengths,optionally through a lens. At least one photodiode detector device (thedevice being an integrated assembly of photodiodes (e.g. photodiodearray), an individual photodiode or compact group of photodiodes) mayalso be included such that the detector is adapted to issue signalsbased upon intensity of light it receives. The instrument may alsoinclude a light transmission assembly that includes at least onemulti-branched waveguide and at least one manifold (e.g., a fiber opticsblock) that is configured to support the waveguide between the sampleholder and both the at least one light emitting diode device and the atleast one photodiode detector device. The waveguide may include a firstfork portion and a second fork portion. The waveguide may include atleast one excitation fork portion and at least one emission forkportion. The first fork portion (e.g., at least one excitation forkportion) may extend between the sample holder and the light emittingdiode device for transmitting light emitted from the light emittingdiode device to the sample contained in the sample holder to excite thefluorescing agent. The second fork portion (e.g., at least one emissionfork portion) may extend between the sample holder and the photodiodedetector device for transmitting light emitted by the fluorescing agentupon its excitation and having a first end that is proximate the sampleholder and a second end that is proximate the photodiode detectordevice. The instrument may include at least one single-band ormulti-band bandpass filter such that the light emitted from the at leastone light emitting diode device is filtered into at least one distinctwavelength band. The instrument may also include a linear variablebandpass filter, a series of bandpass filters, or a multi-band bandpassfilter disposed between the second end of the second fork portion andthe photodiode detector device and is adapted to filter the lightemitted by the fluorescing agent so that the wavelengths of lightreceived across the photodiode detector device are known.

The present teachings further provide for an instrument for performingpolymerase chain reaction with real-time detection comprising apolymerase chain reaction instrument that includes a sample holderconfigured to receive one or more sample tubes that each have at leastone portion that is generally optically transparent, and that receives abiological sample having a nucleic acid to be amplified and at least onefluorescing agent that interacts with the nucleic acid duringamplification and that emits light upon excitation by light of a knownwavelength, in which the signal is generally proportional to the amountof nucleic acid amplified. The instrument may further include a lightemitting diode device that is carried on at least one support substrate,is in electrical communication with a power source, and is adapted toemit light at a plurality of different wavelengths, optionally through acommon lens. At least one heat sink may be included, the heat sink beingcarried on the support substrate for dissipating heat from the at leastone light emitting diode device. At least one photodiode detector devicemay also be included which is adapted to issue signals that aregenerally proportionally based upon intensity of light it receives. Theinstrument may also include a light transmission assembly that includesat least one multi-branched waveguide and at least one manifold that isconfigured to support the waveguide between the sample holder and boththe at least one light emitting diode device and the at least onephotodiode detector device. The waveguide may include a first forkportion and a second fork portion. The waveguide may include at leastone excitation fork portion (e.g., the first fork portion) and at leastone emission fork portion (e.g., the second fork portion). The firstfork portion may extend between the sample holder and the light emittingdiode device for transmitting light emitted from the light emittingdiode device to the sample contained in the sample holder to excite thefluorescing agent. The second fork portion may extend between the sampleholder and the photodiode detector device for transmitting light emittedby the fluorescing agent upon its excitation and having a first end thatis proximate the sample holder and a second end that is proximate thephotodiode detector device. The instrument may also include a linearvariable band pass filter, a series of bandpass filters, or a multi-bandbandpass filter disposed between the second end of the second forkportion and the photodiode detector device and adapted to filter thelight emitted by the fluorescing agent so that the wavelengths of lightreceived across the photodiode detector device are known.

The present teachings also provide for an instrument for performingpolymerase chain reaction with real-time detection comprising apolymerase chain reaction instrument that includes a sample holderconfigured to receive one or more sample tubes that each have at leastone portion that is generally optically transparent, and that receives abiological sample having a nucleic acid to be amplified and at least onefluorescing agent that interacts with the nucleic acid duringamplification and that emits light upon excitation by light of a knownwavelength. The instrument may further include a light emitting diodedevice that is carried on at least one support substrate, is inelectrical communication with a power source, and includes at least four(preferably at least five) light emitting diodes each adapted to emitlight at a different wavelength relative to each other, optionallythrough a single common lens or optic fiber fork. At least one heat sinkmay be carried on the support substrate for dissipating heat from the atleast one light emitting diode device and at least one photodiodedetector device may be included and adapted to issue signals that aregenerally proportionally based upon intensity of light it receives andoptionally including a plurality of discrete pixels or photodiodes. Alight transmission assembly may also be included that is positionedbelow the sample holder and that includes at least one multi-branchedwaveguide and at least one manifold that includes a cavity and isconfigured with flanges to mount to within the polymerase chain reactioninstrument to support the waveguide between the sample bolder and boththe at least one light emitting diode device and the at least onephotodiode detector device. The waveguide may include a first and secondfork portion. The waveguide may include at least one excitation forkportion and at least one emission fork portion. The first fork portionmay extend through the cavity and between the sample holder and thelight emitting diode device for transmitting light emitted from thelight emitting diode device to the sample contained in the sample holderto excite the fluorescing agent. The second fork portion may extendthrough the cavity and between the sample holder and the photodiodearray for transmitting light emitted by the fluorescing agent upon isexcitation and having first end that is proximate the sample holder anda second end that is proximate the photodiode detector device. Thewaveguide may also include a cover portion for the cavity of themanifold that includes a port that is aligned with the light emittingdiode device and an opening for aligning the second end of the secondfork portion with the photodiode detector device. The instrument mayalso include a linear variable bandpass filter that is disposed betweenthe second end of the second fork portion and the photodiode array,wherein the linear variable filter includes a bandpass filter coatingthat is intentionally wedged in one direction, so that the peakwavelength transmitted through the filter varies in a linear fashion inthe direction of the wedge, and wherein the linear variable filter isgenerally optically aligned with predetermined discrete pixels of thephotodiode array, so that the wavelengths of light received by thediscrete pixels of the photodiode array are known upon detection oflight by the array. The instrument may also include a Series of bandpassfilters that is disposed between the second end of the second forkportion and the photodiode array, wherein each bandpass filter of theseries is generally optically aligned with predetermined discrete pixelsor photodiodes of the photodiode detector device, so that thewavelengths of light received by the discrete pixels or photodiodes ofthe photodiode detector device are known upon detection of light by thearray.

As will be seen, the instrument described herein offers a uniqueapproach to providing a modular PCR device providing relativelyhigh-speed PCR amplification and detection by virtue of the device'sability to provide reduced sample read time, and the ability to quicklydetect light at multiple wavelengths. The instrument described hereinmay not rely upon reflective components which are expensive or difficultto align for the routing of light. The instrument described herein maynot rely upon a direct connection from the sample holder and a fiberoptic cable.

DESCRIPTION OF THE DRAWINGS

FIG. 1A is a perspective view of an illustrative real-time cyclingmodule in accordance with the present teachings.

FIG. 1B is a view showing illustrative internal components of the moduleshown in FIG. 1A.

FIG. 2 is an exploded view of the module shown at FIG. 1.

FIG. 3 is an exploded view of an illustrative optical detection modulein accordance with the present teachings.

FIG. 4A is a perspective view of an illustrative fiber optics block.

FIG. 4B is a perspective view of the fiber optics block of FIG. 4A shownwith an illustrative fiber optics cap.

FIG. 5A is a perspective view of the fiber optics block of FIG. 4A shownfrom beneath the block.

FIG. 5B is a perspective view of the fiber optics block of FIG. 5A shownwith an illustrative bottom alignment cover.

FIG. 6 is a perspective view of an illustrative fiber optics cap inaccordance with the present teachings.

FIG. 7 is a perspective view of an illustrative bottom alignment coverin accordance with the present teachings.

FIG. 8 is a top-down view of the fiber optics block of FIG. 4A.

FIG. 9 is a bottom-up view of the fiber optics block of FIG. 4A.

FIG. 10 is perspective view of an illustrative RTD guide.

FIG. 11 is a top-down view of an illustrative circuit board inaccordance with the present teachings.

FIG. 12 is a flow diagram showing an illustrative relationship among thecomponents of an instrument in accordance with the present teachings.

FIG. 13 is a perspective view of an illustrative 4-module instrument inaccordance with the present teachings.

FIG. 14 is a perspective view of an illustrative filter alignment holderin accordance with the present teachings.

FIG. 15 is a perspective view of an illustrative fiber optic block inaccordance with the present teachings.

FIG. 16 is a perspective view of an illustrative sample block/fiberoptic block module in accordance with the present teachings.

FIG. 17A is perspective view of an illustrative tube and cap inaccordance with the present teachings.

FIG. 17B is perspective view of an illustrative tube having an opticallyclear bottom portion in accordance with the present teachings.

FIG. 18 is an exploded side profile view of an illustrative fork portionin accordance with the present teachings.

FIG. 19 is an exploded perspective view of the fork portion of FIG. 18.

FIG. 20 is an exploded side profile and perspective view of anillustrative fork portion in accordance with the present teachings.

FIG. 21 is a side profile view of an illustrative light path of the forkportion of FIG. 18.

FIG. 22 is an exploded side profile and perspective view of anillustrative fork portion in accordance with the present teachings.

FIG. 23 is an exploded side profile and perspective view of anillustrative fork portion in accordance with the present teachings.

FIG. 24 is a bottom view of an illustrative sample tube and opticalfibers in accordance with the present teachings.

DETAILED DESCRIPTION

The explanations and illustrations presented herein are intended toacquaint others skilled in the art with the invention, its principles,and its practical application. Those skilled in the art may adapt andapply the invention in its numerous forms, as may be best suited to therequirements of a particular use. Accordingly, the specific embodimentsof the present invention as set forth are not intended as beingexhaustive or limiting of the teachings. The scope of the teachingsshould, therefore, be determined not with reference to the abovedescription, but should instead be determined with reference to theappended claims, along with the full scope of equivalents to which suchclaims are entitled. The disclosures of all articles and references,including patent applications and publications, are incorporated byreference for all purposes. Other combinations are also possible as willbe gleaned from the following claims, which are also hereby incorporatedby reference into this written description.

This application is related to and claims the benefit of the filingdates of U.S. Provisional Application Nos. 61/681,879 filed Aug. 10,2012 and 61/752,494, filed Jan. 15, 2013. This application is alsorelated to U.S. application Ser. No. 13/484,963 filed May 31, 2012. Thecontents of the aforementioned applications are hereby incorporated byreference for all purposes.

The present teachings pertain generally to an improved device forperforming high-speed real-time polymerase chain reaction. The deviceincludes one or more PCR modules, each PCR module including one or morelight sources, one or more detectors, one or more waveguide devices andoptical componentry for light differentiation. Advantages of theinstrument described herein include reduced componentry which allows forinterchangeability of PCR modules and a reduced footprint. This includesthe ability to employ less hardware per sample. Further, minimalhardware is required per sample such that the functionality of thecomponents described herein is maximized over a wider number of samples.As a specific example, the instrument described herein may require onlyone light source (or one light source component) for multiple samples).Additional benefits provided in accordance with the teachings hereininclude that the optical detection portion is optionally free of movingparts (or moving samples) for increased reliability and fast multiplexdetection. In addition, the present teachings provide for the emissionand detection of multiple colors for multiplex PCR, and for the abilityto give each fluorescent agent an intense specific light color that moreclosely matches the fluorescent agent's peak light absorptionwavelengths. Further, detecting from the bottom of one or more samplesas taught herein may leave side walls of the sample tubes available formaximum heat flow/thermal control and the top of the sample tubesavailable for simplified sample access. The multiple module arrangementof the present teachings also allows for on-demand instrumentavailability and increased sample throughput. The inclusion of multiplesamples per module allows for each sample to be given a nearly identicalthermal profile to better perform statistical comparisons of multiplesamples.

The thermocycler instruments of the teachings herein follow the basicprinciples of WO/2009/105499 and U.S. application Ser. No. 12/918,594(U.S. Publication No. 2011/0039305) and Ser. No. 13/484,963 in that asample block (e.g., a sample holder) is sandwiched between opposingthermoelectric devices. The teachings, however, address a number of newfeatures for thermocycler instruments that successfully and unexpectedlyimprove efficiency and operation of the instruments as compared withinstruments that do not employ such features. The teachings furtherprovide for thermocycler instruments that facilitate simultaneousamplification and quantification of nucleic acids.

The nature of the sample block being sandwiched between opposingthermoelectric devices indicates that samples located within the sampleblock receive light from a light source from either above or below thesample holder, given the difficulty with transmitting light through thethermoelectric devices. As a further result of the sandwich design,detection should preferably also occur from above or beneath the sampleholder. It is also possible that the fiber optics may be integrated intothe sample block in which excitation and/or emission light istransmitted through the side of the tube. It is also possible thatapertures may be present within the thermoelectric devices such that theoptical pathway for emission and/or detection occurs through theseapertures.

The instrument thus includes a combination of components selected andadapted for relatively rapid cycling and real time quantification ofnucleic acids. For example, the teachings herein contemplate use ofinstruments within the teachings for performing real-time nucleic acidamplification over a period less than about 40 minutes, less than about30 minutes, less than about 20 minutes, or even less than about 15minutes. The teachings herein contemplate that such amplification andquantification rates can be successfully employed for sample sizes ofgreater than about 2 microliters, 10 microliters, 30 microliters, 50microliters, or even 70 microliters (e.g., a sample size of about 25microliters). Relatively large yields of amplified nucleic acids (e.g.,at least levels detectable by gel electrophoresis) are possible over arelatively short period of time. The unexpected ability to perform suchrapid real-time amplification and quantification on relatively largesample sizes is one of the advantageous aspects of the presentteachings. Aspects of the instrument also may be premised upon therecognition that thermal inertia characteristics of structures andmaterials used for thermocycler instruments can impede the rate at whichthermal cycling can take place as may intrinsically occurring lags thatoccur due to electronic processing capabilities of an instrument.Accordingly, the present teachings also pertain, in various aspects, tounique approaches addressing such obstacles.

The teachings herein envision the efficient operative employment of atleast one first thermal cycling element for thermally cycling a samplein generally opposing relationship with at least one second thermalcycling element for thermally cycling the sample. Though other devicesmay be employed, the thermal cycling element for thermally cycling thesample typically will be one or more thermoelectric devices (“TEDs”).Thus, it is envisioned that a first TED and a second TED may be ingenerally opposing relation with one another. They may be generallyidentical and may be controlled to operate substantially identicallywith each other. A sample holder may be employed to carry a sample(e.g., a sample enclosed or otherwise carried within a sample container,such as a tube). The sample holder may be adapted to receive at leastone sample (e.g., a sample carried in a sample container such as a tube)and to be disposed (e.g., in a sandwiching relationship) between thethermal cycling elements (e.g., between the first TED and the secondTED). The sample holder may be adapted to receive a plurality ofsamples. Further, the thermocycling instrument may include a pluralityof sample holders, each sample holder being located in thermalconducting relation with one or more thermal cycling elements. Thethermal cycling elements may each include at least one heat exchanger(e.g., a suitable heat sink) for transferring heat relative from or toeach of the cycling devices (e.g., the first and second TEDs).

The sample holder may be formed within a sample block, which may bemetallic sample block (e.g. heat block). The sample block may be asilver sample block. The heat block to house samples may be manufacturedfrom rolled silver stock that has the oval bores formed by wireelectrical discharge machining (EDM). An alternative manufacturingprocess such as a casting process or potentially splitting the blockinto two pieces for direct machining may also be used. Additionally, aslight increase in sample block thickness (e.g., an increase to about 3mm) may improve temperature uniformity among the bores that receive thesamples while having minimal impact on speed of the thermocycling.

As with other hardware components described herein, the above componentsmay be configured and positioned in a way to afford efficient heattransfer to and from a sample. They may be configured and positioned ina way to help potentially reduce thermal inertia obstacles to efficientheating or cooling. They may be configured and positioned in a way toachieve substantially uniform heating or cooling rates to a plurality ofsamples that may be carried within the sample holder. Thus, the firstTED and second TED may both operate to heat and/or cool one or moresamples simultaneously in an effort to achieve a more uniform heatingand/or cooling. They may be configured and positioned in a way tosubstantially account for the thermal phenomena of the components inexecuting temperature control.

To improve temperature control a fan may be included within theinstrument. The fan may be a variable speed fan. The incorporation of avariable speed fan is advantageous in that it reduces audible noise andmay also provide improvement to temperature uniformity at low fanspeeds. Although, some PCR protocols may be run with no fan. The fan maybe powered by a signal that is pulse width modulation (PWM) controlled.Thus, a fan may be selected that can handle a PWM power signal, oradditional electronic circuitry may be added to modify a PWM signal intosteady DC voltages.

The teachings envision the use of stable temperature sensingcomponentry. For example, the componentry may be such that two or moresensors, which may include two or more sensors for each sample block,monitor (e.g., simultaneously and/or continuously) at least twotemperature conditions, each originating in a location remotely of eachother within the thermocycling device. For example, one sensor mightsense a condition within the above-noted sample holder that approximatesthe temperature to which the sample is being subjected. That is, thetemperature conditions are selected so that they can be relied upon asbeing related to (e.g., generally corresponding with in a direct manner)the temperature within the sample holder, which may be indicative of thetemperature of a sample located within the sample holder. Another sensormight sense a temperature condition of a component, such as a heatexchanger, that provides useful information in accounting for thethermal inertia and thermal interactions during heating, cooling, andtemperature holding. The componentry also is adapted to deliverinformation (e.g., via electrical signaling) corresponding with one ormore sensed temperature conditions.

The teachings also contemplate a method (e.g., a computer-implementedmethod) for operating a thermocycler (such as the thermocyclers asdescribed herein) for real-time amplifying and quantifying of nucleicacid (e.g., DNA (deoxyribonucleic acid)) of a sample (e.g., a patientsample, such as a human patient sample). The method may includeobtaining user input such as in the design of a desired PCR protocol.The method may include a step of displaying one or more user interfaces.Such user interfaces may be configured so that a user is able to inputoperational instruction protocol information for operating thethermocycler. Such instruction information may be selected from one orany combination of at least one temperature setting, number of cycles tobe performed, times for one or more cycles, hold times at one or moretemperatures, or the like. The method may include a step of receivinguser operational instruction protocol information inputted by the user.The method may include a step of causing the thermocycler to execute aprotocol for nucleic acid amplification and/or quantification on thebasis of the operational instruction protocol information inputted bythe user. The method may include a step of storing (e.g., in a suitablememory device in communication with the instrument) operationalinstruction protocol information inputted by the user. The method mayinclude a step of displaying for a user previously programmedoperational instruction protocol information so that the user can modifysuch information for designing a protocol. The method may include a stepof receiving information about a temperature condition to which a sampleis being subjected and causing such information to be displayed to auser substantially in real time. The method may include a step ofproviding a user with an opportunity to start, stop and/or pause aprotocol during execution of such protocol on a sample. The method mayinclude a step of outputting data about an actual or proposed protocol.The method may include one or any combination of other steps of storingnotes Inputted by a user, providing a preview of a protocol beforecausing a thermocycler to execute the protocol, or performing adiagnostic check to ascertain operability of a thermocycler.

The method may include a step of receiving optical input parameters fromthe user which includes one or more of the following: the PCR cycles inwhich to perform an optical read, the fluorescing which are being usedfor the optical detection, the color wavebands to use to excite thefluorescing agent, the color wavebands to use to detect the fluorescingagent, the sample identification, the sample type (no sample, knownstandard, unknown sample, positive control sample, negative controlsample, or no DNA template control sample), the DNA template quantity ofknown standards and/or controls, and melting curve information includingtemperature start, temperature stop, and melting curve resolution. Themethod may include a step of storing the optical input parameters. Themethod may include a step of retrieving stored optical input parameters.The method may include a step of displaying optical detection data whichincludes one or more of the following: compatibility of user-selectedfluorescing agents, estimated quantification threshold (e.g. thefractional cycle number at which the amplified DNA is detectable fromthe background noise), estimated initial DNA copy number for unknownsamples, estimated melting temperature of the detected DNA. The methodmay include a step of graphing the optical detection data which includesone or more of the following: a graph of the optical signal as afunction of PCR cycle number, a graph of the optical signal as afunction of sample or sample-holder temperature, a graph of the negativeslope of the optical intensity with respect to temperature as a functionof temperature, a graph of the actual and/or expected optical data as afunction of wavelength. The method may include a step of storing theoptical data. The method may include a step of retrieving stored opticaldata. The method may include a step of applying the temperature and/oroptical input to one or more modules independently or simultaneously.The method may include a step of displaying, storing, or retrievingstored data from one or more modules. The method may include step ofobtaining temperature and optical input from a barcode scanner, 2Dbarcode scanner, NFC (near field communications), or RFID (radiofrequency identification) from an appropriate test kit, assay, or sampletube.

The teachings herein also contemplate a non-transitory computer readablemedium comprising program instructions for performing the methods (orany of the steps) as described in the herein. The teachings thusenvision at least one computer software or firmware program includingcode that provides instructions to hardware for performing PCR which,when executed by a suitable electronic processor or other computerprocessor, performs the methods (or any of the steps) as described inthe herein. The teachings also contemplate a system for performing a PCRamplification reaction the system comprising a device including a memorystorage medium for implementing the program instructions of thenon-transitory computer-readable medium. The memory storage medium maybe on a computer (e.g., a computer having a processor with a processingspeed of at least about 1.67 GHz, such as an Inspiron Mini 1018, fromDell). The computer may be external and attached to the instrument.computer may be internal to the instrument (e.g. an industrial mini-ITXbased computer with extended life cycle such as a J&W ITX-IC2M1026S(available from J&W, South San Francisco, Calif.) dual core atommini-ITX mainboard) with a display unit (e.g. a screen or atouch-screen) internal to the instrument or a port for attaching anexternal monitor. The computer may employ a Windows®-based operatingsystem, or some other like system. The system may include a thermocyclerin accordance with the present teachings, one or more devices forcollecting information about the temperature condition of the sampleholder, and an output device for displaying data obtained or generatedby the analyzing device. The output device may be a display panelassociated with the computer. Multiple functions of the software may becaused to be performed by code on a single non-transitory storagemedium, or on multiple media. For example, functions may reside onfirmware associated with a controller that is on-board the thermocyclerinstrument.

The instrument of the present teachings may require the use of aspecialized tube for facilitating qPCR (quantitative real timepolymerase chain reaction) that facilitates transfer of light. The tubepreferably allows for detecting the emission of light from fluorescentdyes/probes within the tube. The amount of light detected is generallyproportional to the amount of formed PCR product, resulting insimultaneous PCR-based amplification and detection. Preferably, the tube(or at least a portion thereof) is optically clear for high transfer oflight to/from a reaction mixture within the tube with at least about 50%transparency (and preferably at least about 80% transparency) to thevisible light spectrum.

As an example, an optically clear resin may be used and injection-moldedto form the entire tube. An exemplary polypropylene resin is a cyclicolefin copolymer resin from TOPAS Advanced Polymers, sold under thetrade name 5013S-04. Another exemplary polypropylene resin is aclarified, high-melt random copolymer from LyondellBasell Industries,sold under the trade name Pro-fax RP448S. These resins exhibit highflowability to fill thin-walled areas pf the tube while providing hightransparency.

As a further example, the tube may be optically transparent only inportions of the tube in which the light excitation and emission occurs.Both excitation and emission may be performed throngh the bottom of thetube and thus only the bottom tip of the tube need be optically clear.As such, a two-shot mold process may be used to create a bottom portioncomprised primarily of an optically clear resin with the rest of thetube may comprise a second, different resin that is not necessarilyoptically clear.

An over-molding technique may also be used to construct an opticallyclear portion of the tube. By way of example, an optically clear bottompiece may be over-molded during the tube fabrication process (see FIG.17B). The optically clear piece may be introduced into the mold cavityprior to injection of a polymeric material. The optically clear portionmay be flat to minimize light reflection, light distortion, and lightabsorption by the sample tube. The optically clear portion may be a lensshaped plastic, glass, fiber optic cable, or the like. The opticallyclear portion may also serve to stabilize the core-pin of the injectionmolding process to minimize core pin deflection.

Alternatively, a secondany external operation may be utilized to form anoptically clear portion of the tube. By way of example, the tube may bemolded with an open end to which an optically clear piece is theninserted. Fusion between the tube and the optically clear piece may beachieved by thermal bonding, adhesive bonding, mechanical fitment, orother means as known in the art of plastics bonding and manufacturing.

One or more optically clear portions of the tube may take on specificdimensions and curvatures (i.e. lens design) to achieve optimal coupling(i.e. transmission, dispersion, and focusing of light) among the samplefluid, the excitation means, and the detection means. The opticallyclear portions may be comprised of planar, concave, convex, meniscus,Fresnel, or other lens surfaces as known in the art of optics. In apreferred embodiment, the shape of the optical pathway of the bottom ofthe tube may be comprised of two substantially parallel planar surfaceswith a relatively thin wall thickness. A high degree of polish of theoptical pathway surfaces is also advantageous to light transmission.

The one or more optically clear portions may be formed on the bottom ofthe tube. Alternatively, the one or more optically clear portions may beformed along the top of the tube. The top of the tube (i.e. cap) may beoptically clear in embodiments in which excitation, emission, or bothoccur above sample fluid (see FIG. 17A). In one embodiment, the lightsource may be located above the sample fluid while the detection meansis located below the tube, such that the tube may include more than oneoptically clear portion. In another embodiment, both the light sourceand detection means are located above the tube such that transmission oflight necessary to conduct real-time PCR occurs through the cap.

As mentioned above, a light source may be utilized within theinstrument. The light source may be located within the instrument suchthat it provides light through one or more optically clear portions of atube in which a sample is located. The light source may be located on aprinted circuit board. The printed circuit hoard may thus provide anelectrical supply to the light source. The light source may include oneor more light emitting diodes (LEDs). In the event that the instrumentcontains more than one sample block, each sample block may include itsown light source. Each sample block may have multiple light sources,with one or more light sources for each sample well or a shared lightsource among wells (e.g. one light source optically connected to two ormore sample wells). Each light source may be carried on a commonsubstrate. Further, each light source may include a plurality ofdistinct lights such that each distinct light provides light at adifferent wavelength. As an example, each sample block may include anarray of LED lights, each array including distinct lights at one, two,three, four, or more different wavelengths in order to better match thepeak optical absorption wavelengths of various fluorescent agents. Inthis case, the LED light sources may be grouped underneath a fiberopticwaveguide such that one or more light sources enter the same fiber opticfork. Alternatively, the LED's may be grouped together on the samecommon substrate (i.e. a compact printed circuit board or assembly), butwith each LED positioned beneath its own fiber optic fork. A pluralityof high power LEDs (of wavelengths typically covering the 400 nm to 700nm visible light region) may be grouped together in an area less thanabout 3 mm by 4 mm (an example of which is available from PhilipsLumileds Lighting Company under the designation Luxeon Z). One suchgrouping may include four Luxeon Z LEDs with wavelength peaks ofapproximately 477.5 nm, 522.5 nm, 585.5 nm, and 665.0 nm. A second suchgrouping may include four Luxeon Z LEDs with avwavelength peaks ofapproximately 447.5 nm, 494.0 nm, 537.5 nm, and 635.0 nm. Two or moresuch groupings may be incorporated in each module with each groupinghaving its own fork of the fiber optics waveguide and optionally its ownmulti-band bandpass filter.

Alternatively the grouping may include seven Luxeon Z LEDs which arestaggered to form a hexagonal shape and include up to seven colors withwavelength peaks selected from the following list: 442.5 nm, 447.5 nm,452.5 nm, 457.5 nm, 462.5 nm, 467.5 nm, 472.5 nm, 477.5 nm, 494 nm, 503nm, 522.5 nm, 527.5 nm, 532.5 nm 537.5 nm, 585.5 nm, 588.5 nm, 591 nm,593.5 nm, 615 nm, 625 nm, 635 nm, 655 nm, or 665 nm. A grouping of sevenLEDs staggered to form a hexagonal shape may include wavelength peaks ofapproximately 477.5 nm, 494 nm, 522.5 nm, 537.5 nm, 585.5 nm, 635 nm,and 665 nm. The groupings of LEDs may have a lens to focus light throughone or more filters and/or into one or more fiber optic excitationforks. The lens may be an array of individual lenses or it may be asingle unit with multiple integrated lense, one for each LEO.Alternatively, each LED light source may include only 1 distinct lightadapted to emit a plurality of different wavelengths. In this case, aplurality of LEDs (each of a different peak wavelength) may beencapsulated behind a single lens within a single assembly (an exampleof which is available from LED ENGIN, Inc., under the designationLZ4-00MA00). Each compact grouping or single assembly of LEDs may beconsidered as a light emitting diode device. As alternatives to lightemitting diodes, white light sources such as halogen or tungsten bulbsor lamps, laser light sources, or other excitation means may beemployed.

the light source may be part of an assembly that includes a carrierhaving a first surface and a generally opposing second surface. Thelight emitting diode may be exposed via the first surface. One or moreelectrical contacts (e.g., pads) may be located on or as part of thesecond surface and be in electrical communication with the diode. Inthis manner, the pads may be applied to a substrate (e.g., by way of asoldering to a printed circuit board). The upper surface may invlude oneor more apertures through which the light may be emitted from the LEDs.The upper surface may include one or more conduits of a predetermineddepth (e.g. about 1 mm, 2 mm, 3 mm, 4 mm, 5 mm or higher) that may besuitably adapted to connect in light transmission relationship with awave guide structure (e.g., a fiber optic structure). The conduits maybe elongated and include a longitudinal axis. They may be generallycylindrical. They may be at least partially conical. They may include agenerally round, oval, triangular, rectangular or other polygonalcross-sectional profile relative to the longitudinal axis. They may havea wall structure defining a passage in the conduit that has a taper(e.g., less than about 15, 10, or even 5°, though tapers of at least 20,30 or 45° are possible) relative to the longitudinal axis.

The light source will typically include an exposed end through whichlight is emitted. For each light source of a predetermined wavelength,the end may have an area that is smaller than about 9 mm², 6 mm², oreven 3 mm². It may have an area that is larger than about 0.5 mm², 1mm², or even 2 mm². The emitted beam may have an emission axis, and mayexhibit a generally linear, rectangular, oval, circular, or othercross-sectional profile relative to the emission axis.

The light source may exhibit one or any combination of performancecharacteristic as set forth in the LUXEON Z Datasheet DS105 20120916,incorporated by reference herein (without limitation, pages 3 through 9,page 14-20, and 24 through 27). The light source may exhibit one or anycombination of structural characteristic as set forth in the LUXEON ZDatasheet DS105 20120916, incorporated by reference herein (withoutlimitation, pages 10 through 13 and 21 through 23).

The light source may be a relatively high power light source which mayprovide for more sensitive detection capability. As an example, thelight source may be rated at 40 Watts or more, although the light sourcemay or may not be operated at the maximum level. As a result of the highpower of the light source, it may be capable of dissipating heat. Thelight source may thus be in close thermal communication with a heatsink, which may be located onto the printed circuit board. The heat sinkmay be located beneath, and/or around, the light source. The heat sinkmay assist in dissipating heat from the light source.

An additional benefit of LEDs is that they use less power than othertypes of light sources (e.g., compact fluorescent or incandescent bulbs)per unit of light generated. LEDs also have improved durability ascompared to other light sources. In addition, the use of LEDs as thelight source allows for compact packaging for insertion into smallspaces within the instrument. Preferably the packaging for the lightsource may be less than 3 cm on each side, or less than 1 cm on eachside, or even less than 0.8 cm on each side or a grouping of LEDs withthe grouping being less than 1 cm on each side, or even less than 4 mmon each side. As a result, LEDs allow for effective output andperformance from a device that occupies minimal space. In oneembodiment, the light source can be located beneath the heat exchangers.In an alternative embodiment, the light source may be located above thesample block. In the event that the fiber optics are flexible, the lightsource may be located anywhere depending upon the arrangement of thesamples and the nature of the tubes containing the samples. The smallpackaging of the light source assists in maintaining the small,lightweight and portable nature of the instrument.

The selected light source should be compact, compatible with a fiberoptics design, and sufficiently bright. In the event that LEDs areselected as the light source, it may be beneficial for multiple LEDelements to be located into a single housing. For example, a singlehousing may include at least 4, at least 5, at least 8, or even at least12 LED elements such as the LuxiGen family of LEDs available from LEDEngin, San Jose. Calif. Any LED lens may be formed with a flat top forimproved connection to any fiber optic cable. Ultra-small LEDs may beutilized such as Luxeon Z LEDs, Phillips Lumileds Lighting Company, SanJose, Calif. or XLamp LEDs from Cree, Morrisville, N.C. Theseultra-small LED's may be compactly grouped together. A four-color LEDgrouping may be utilized as the light source. A seven-color LED groupingin a hexagonal shape may be utilized as the light source with sevendistinct colors. A seven-color LED grouping in a hexagonal shape may beutilized as the light source with five distinct colors and one or twocolors repeated for additional light intensity of that specific colorand/or additional lifespan by switching from one LED of a specific colorto another LED of that same color. An eight-color LED combination may beutilized as the light source.

The instrument may also include a device for detecting a reaction withina sample. The detector may include a photodiode array which issues asignal proportionally based upon intensity of light it receives. Anexample of a photodiode is the Taos TSL 1402R, available from AMS-TAOSUSA Inc., Plano, Tex. The detector may be located within less than about10 mm, less than about 5 mm, or even less than about 3 mm from an end ofa waveguide to help avoid light from becoming diffuse. The detector maybe located in an isolated contained chamber so that it is not exposed toany other light source and is insulated from heat generated by the restof the instrument. The chamber may be formed as a surrounding wallstructure that substantially insulates the detector from other light.The detector may be formed as an individual array for each sample oralternatively may be a singl array subdivided into array portions thatare dedicated to individual samples. The detector may be formed asarrays arranged in elongated thin strips so that pixels of the arraysare aligned end to end. Each elongated strip may include from about 25to about 200 pixels (each being about 65 microns by 55 microns). Thedetector may be a two-dimensional array of pixels such as withcomplementary metal-oxide-semiconductor (CMOS) or charge-coupled device(CCD) detector circuitry. Alternatively the photodiode array may consistof several larger individual photodiode elements (about 1 mm×1 mm, orabout 3 mm×3 mm) each with one pixel per detection color. Aternatively,PIN (p-type, intrinsic and n-type semiconductor regions) photodiodes maybe employed and may be present with one PIN photodiode per sample,multiple PIN photodiodes per sample, or one PIN photodiode shared bymultiple samples. An array of the PIN photodiodes may exist for eachsample in sitruations where each PIN photodiode is paired with abandpass filter of a specific wavelength. For instances in which one PINphotodiode is used per sample, a lens may be used which directs lightfrom at least one bandpass filter to the single PIN photodiode. Forinstances in which one PIN photodiode is used by multiple samples, alens may be used which directs light from at least one sample to the PINphotodiode.

Each array may monitor one, two, three, or more samples at a time. Eacharray may be adapted for moving from sample to sample. The arrays may bearranged to read more than one pixel at a time (from more than onesample). The time between the readings may affect sensitivity due to theentry of light. It may thus be desirable to complete readings as quicklyas possible (e.g., less than about 0.5 milliseconds average per pixelper reading) to maximize sensitivity. It may be desirable to completereadings for each pixel less than 0.1 second, or even less than 0.01seconds. A sum of reads for each pixel may be used to result in a totalread value per specified integration time. It may be desirable tocomplete the total reads of all samples for all dyes in less than 10seconds, less than 5 seconds, or even less than 3 seconds to maintainminimal run times of the instrument.

In another embodiment, the photodiode array may simply be a grouping ofindividual photodiodes. In this case, unique bandpass filters may bepositioned above each photodiode such that the signal from thatphotodiode is related to a specific fluorescent agent. In yet anotherembodiment, a single photodiode (e.g. PIN photodiode) is used fordetection of all fluorescent agents. In this case, different bandpassfilters may be moved into position above the photodiode (e.g. a filterwheel or shuttle) so that the signal generated at a specific timecorresponds to a fluorescent agent. This allows for detection ofmultiple fluorescent agents by cycling through the filters during adetection step. Alternatively, a stationary multi-band bandpass filtermay be employed above the photodiode such that the light detectedcorresponds to a certain fluorescent agent, dictated by the excitationwavelength provided at that instance.

Alternatively, the detector may include at least one spectrometer whichmay also require the use of a prism device or optical diffractiongrating to separate light according to different wavelengths. Thedetector may also include at least one charge-coupled device or othercapacitor containing device or photomultiplier tube.

The ability to excite one or more probes contained within a sample fortesting may be enhanced by employing one or more features forcontrolling the light that is directed to the sample holder from one ormore light sources. For example, without limitation, as to light fromone or more light source, one or more features may be employed toattenuate, intensify, modulate, collimate, refract, reflect, diffract,or filter such light or any combination of the foregoing.

Consistent with the foregoing, the ability to detect light from one ormore excited probes contained within a sample may be enhanced byemploying one or more features for controlling the light that is emittedfrom the sample (or at least one probe therein). For example, withoutlimitation, as to light from a sample, one or more features may beemployed to attenuate, intensify, modulate, collimate, refract, reflect,diffract, or filter such light or any combination of the foregoing.

An approach that may be employed for enhancing transmission of light forexcitation of one or more probe, for detecting fluorescence emitted byone more probe or both may involve the selection of a suitable filterarrangement. One or a combination of two or more filters may be employedfor this purpose. Selection of a filter for this purpose may be basedupon one or more desired attribute of the filter.

In the context of detecting light, it may be expected in some instancesthat a filter is selected by which a significant amount of light of oneor more predetermined wavelengths is allowed transmission through thefilter for affording a larger amount of detectable light for a detector.For example, it may be possible that one or more absorptive filter isemployed, such as a filter with an optical density (OD) value of about4, 3, 2, 1 or lower. Successful results may be achieved by the use ofone or more filters having an OD value of greater than 4 (e.g., a valueof OD 5, OD 6, or OD 7). The cumulative OD value of such filters may begreaterthan 4 (e.g. a value of OD 5, OD 6, or OD7). The OD values arebased upon transmission values measured at a wavelength from about 400nm to about 800 nm in accordance with spectrometer according to standardoptical metrology transmission measurement techniques (often a custommodified spectrometer is used to measure large optical densities, overabout OD 4, and to measure filters with sharp transitions in opticaldensity as a function of wavelength).

The filters may be present on the LED waveguide fork, the detectionwaveguide fork, or both. The filters may be separate components or thefilters may be deposited directly onto lenses, LEDs, photodiode detectordevices, and/or fiber optic waveguides.

The filters may be neutral density filters. They may be uncoated. Theymay be metallic coated. They may be made of optical quality glass,UV-grade quartz or some other suitable material.

One or more interference filters may be employed for selectivelyallowing transmission of light within one or more predetermined range ofwavelengths, while reflectin light of other wavelengths. For example,one or more dichroic filters may be employed. Examples of suitabledichroic filters may exhibit one or more performance characteristicsincluding transmitting light from the LEDs at the excitation wavelengthrange(s), and reflecting light at the fluorophore emission wavelengthrange(s) (or the reverse of reflecting the excitation light andtransmitting the emissed light). An example of a suitable dichroicfilter employed herein is commercially available from Edmund Optics,Barrington, N.J. under the designation #67-055.

One of more filters may be employed at one or locations within a system.One or more filters may be employed between a source of light and awaveguide (e.g., a fiber optic structure) through which the light istransmitted. One or more filter may be employed between a light emittingportion of the waveguide (e.g., fiber optic structure) and the sample(and/or holder within which the sample is ontained). One or more filtermay be employed between the sample (and/or holder within which thesample is contained) and any detector.

One or more components of the system may have a filter assembled to it.One approach may be to select materials for the sample holders of thesystem by which the material intrinsically filters one or morepredetermined wavelength or range of wavelengths.

One example of a filter that may be employed herein is a linear variablefilter. For example, such a filter may be employed in advance of adetector of the system. Another option that may be employed alone or incombination with a linear variable filter may be to employ one or morebandpass filters or other filter. Examples of suitable bandpass filtersmay exhibit one or more performance characteristics including a hardcoating, at least 90% transmission in the bandpass wavelength range, anoptical density of at least OD5 in the blocking wavelength ranges, atransmission band of approximately 10 nm to 50 nm, and a sharptransition (less than about 5 nm) between the transmitting wavelengthsand the blocked wavelengths. Example of a suitable bandpass filteremployed herein is commercially available from Edmund Optics,Barrington, N.J. under the designation 67-013.

Any linear variable filter may be utilized for filtering light such thatonly light having certain wavelengths can pass through the filter atdifferent filter locations. As a result, only light of a knownwavelength may pass through the filter and to the detector (e.g.,specific pixels of an array) so that the light that is passing throughis a predetermined known wavelength for which only intensity needs to bemeasured for each pixel in the detector. Examples of suitable linearvariable filters may exhibit one or more performance characteristicsincluding a hard-coating, separation of light to a spectral range fromabout 450 nm to about 800 nm, average transmission of over 40%, and anoptical density of at least OD3. Examples of suitable linear variablefilters employed herein are commercially available from Delta, Hørsholm,Denmark under the designation LF102155 or JDS Uniphase, Santa Rosa,Calif. under the designation 30119150. The filter may be substantiallysimilar in size to the detector. The filter may be located onto asupport with the detector or may be located on a separate support fromthe detector. The filter may be permanently adhered to the detector withoptically transparent adhesives so that there is a precise andrepeatable relationship between the wavelength and pixel number. Whiletypically the transmitted wavelengths will vary linearly across thelinear variable filter, other monotonic functions (e.g. logarithmic) maybe utilized.

As an alternative to the linear variable filter, a series of discretebandpass filters may be deployed. The series of bandpass filters mayinclude distinct filters grouped together or an integrated assembly inwhich various portions of a substrate represent different bandpasscharacteristics. The bandpass filters may be lined in parallel so thatthe assembly aligns optically with the detector pixels or photodiodes.In this respect, this embodiment can be simply viewed as a linearvariable filter with discrete step-wise portions rather thancontinuously variable. Alternatively, a multi-band bandpass filter maybe used in conjunction with the detector. In this case, the wavelengthof light transmitting through the multi-band bandpass filter to thedetector is related to the excitation light provided at that instancebased upon the excitation/emission behavior of the fluorescent agent.The discrete bandpass or multi-band bandpass filters may be arranged ina fixed position generally with one filter per LED or photodiodedetector device. The discrete bandpass or multi-band bandpass filtersmay be arranged on a filter-wheel which moves the excitation and/ordetection filters between the LED and fiber optics and/or between thefiber optics and the photodiode detector device.

Another example of a filter system would be a aeries of dichroricbeamsplitters, dichroric filters, dichroric mirrors, and/or dichroicprisms which reflect light of certain wavelength ranges at anapproximately 90° angle to the incoming light and the reflected coloredlight branches then pass through a series of suitable bandpass filterslocated over a detector device. In this latter example, the incomingbeam of light is split into one or more different colored light branchesby passing through or being reflected by the series of dichroricbeamsplitters. The series of bandpass filters further filter the coloredlight branches into specific wavelength ranges. Each bandpass filter islocated over a known number of photodiode elements in a photodiode arrayand/or individual photodiodes in a compact group of photodiodes withinthe detector device. Thus, the intensity of light of each colored lightbranch can be measured simultaneously with no moving parts. An exampleof a suitable colored light branch detection device is the OptoFlashOptical Engines from Newport Corporation, Franklin, Mass. One or moreoptical fibers can be used within the same OptoFlash, and thus multiplesample tubes can be ansyzed sequentially by exciting each sample tubesequentially with light. A custom OptoFlash may be used with filteredcolor light branches with light wavelength ranges of approximately 510nm to 547 nm; 555 nm to 565 nm; 565 nm to 575 nm; 575 nm to 600 nm; 608nm to 655 nm; 665 nm to 691 nm; and 699 to 770 nm. Additional colorlight branches could be added to the OptoFlash to detect additionalwavelength ranges or fewer color light branches. The color light branchwavelengths could be adjusted to be more suited for differentfluorescing agents with the color light branch bandwidth being used tocontrol sensitivity and to control the total number of differentfluorescing agents that will be detected.

The employment of one or more suitable waveguide structures may beemployed as discussed herein. One particular approach is to employ afiber optic structure. Though it may be believed that the selection andemployment of a suitable fiber optic structure is readily within theskill in the art, experience is believed to prove the contrary. Withoutintending to be bound by theory, it is believed that the selection of asuitable optical fiber structure may not be readily predictable. By wayof example, it is possible that certain applications may seek to employa fiber optic structure having a relatively large numerical aperturevalue (e.g., greater than about 0.55, 0.65 or even 0.75). This may be asensible approach both for assuring a relatively large transmission oflight for excitation, a relatively large angle of light acceptance(e.g., at least about 33°) or both. However, for sensitivities desiredfor certain of the applications herein, such structure may actually leadto less efficient performance in the way of light transmission and/ordetection. Thus, the teachings herein contemplate the employment offiber optic structures that have a numerical aperture value that is lessthan about 0.52, 0.50, 0.45, 0.40 or smaller (e.g., to as low as about0.5).

The structure may be configured so that the angle of light acceptancemay also be a value that is below about 32%, 30%, 25%, 20% or lower. Itmay be an angle of 5%, 10% or higher. The angle of incidence is measuredas the angle between the normal surface (of the terminal end of theoptical fibers) and the incident light ray.

It will be recognized that numerical aperture values refer generally toa maximum angle at which a particular fiber can accept the light thatwill be transmitted through it. The numerical aperture value of anoptical fiber is thus correlated with the size of a cone of fight thatcan be coupled into its core. As the skilled artisan will appreciate, avalue for numerical aperture can be derived by calculating the sine of ahalf angle of acceptance within a cone of light that enters a core of afiber. An approach to measure numerical aperture is illustrated in TIAFiber Optic Test Procedure FOTP-177 (Method Numerical ApertureMeasurement of Graded-Index Fiber.

The waveguide may be a bifurcated waveguide such that it includes afirst and second fork portion. The first fork portion may extend betweenthe sample holder and the light sourrce (e.g., an LED device) fortransmitting light emitted from the light source to the sample containedin the sample holder to excite a fluorescing agent. The second forkportion may extend between the sample holder and the detector (e.g., aphotodiode detector device) for transmitting light emitted by thefluorescing agent upon its excitation and having a first end that isproximate the sample holder and a second end that is proximate thedetector. The waveguide may be formed of a single structure or may beformed of optical fiber bundles. The waveguide or waveguide fibers maybe formed of a polymeric or glass material. The waveguide or waveguidefibers may be made of single-mode optical fibers or multi-mode opticalfibers, or any combination thereof. The waveguide may have the opticalfilters (bandpass filters and/or linear variable filters) or lensesdirectly deposited on the terminal ends.

The waveguide may be a multi-branched waveguide such that it includes atleast one excitation fork portion (e.g., a first fork portion) and atleast one emission fork portion (e.g., a second fork portion). Thewaveguide may include at least two, at least three, at least four, atleast five, or more excitation fork portions. The number of excitationfork portions may be the same as the number of LED's present in thelight emitting de device. The excitation fork portion end proximate theend of a light source may branch out to provide light to one, two, four,eight, or more samples. The waveguide may include at least two, at leastthree, at least four, at leave five, or more emission fork portions. Thenumber of emission fork portions may be the same as the number ofdetector wavelength ranges. The at least one emission fork portionproximate a sample may branch out to different detector portions. The atleast one emission fork portion proximate a detector or detector portionmay branch out to provide deteotion of at least one, two, four, eight,or more samples. In embodiments in which the excitation occurs fromabove the samples, the waveguide positioned below the samples may bevoid of excitation fork portions with only emission fork portionspresent.

The waveguide may be arranged so that a terminal end interfaces with thedetector and will be elongated to coincide with the elongated structureof an array as discussed above. The instrument may include a manifoldassembly that connects with the printed circuit board that carries thelight source (e.g., the LEDs), and includes passages. These passages mayallow for isolation of the individual light source assemblies and may beadapted to receive the waveguide (e.g., fiber bundles).

The instrument may include a housing for receiving the waveguide. Thehousing may include an upper portion that is adapted to fit in betweenthe heat exchangers and to be aligned with (and located below) a sampleholder. The waveguide may partially extend into the wells of the sampleholder for close coupling and alignment of the waveguide to the sampletube. The housing may include one or more projections for aiding inaligning the housing within the instrument. The housing may also includeone or more mounting flanges to provide a surface for attaching to acavity within the instrument. The housing may further include a baseportion having a cavity defined therein through which one or both forkportions of the bifurcated waveguide (e.g., fiber optic bundles) arepassed, and which can receive a resin for potting the waveguide. Thehousing may also include one or more cover portions. A bottom coverportion may be adapted to interface with the detector and may be locatedabove the printed circuit board and light source located thereon. One ormore ports may also be formed along a surface of the housing so that theone or more ports align with the light source. The light source maypenetrate through the ports or alternatively may remain adjacent to theports without penetrating the ports. There may be an optical filter(such as a bandpass filter) between the light source and the penetratingports.

The detector may be adapted to receive light from a plurality ofsources. For example, the detector may receive (e.g., detect) light froma fluorescing sample and light reflected from the light source. Multiplefluorescing agents with different emission wavelengths may be present inthe sample. As such, it may be necessary for the detector to be capableof differentiating different colors (e.g, light emanating from differentsources and fluorescing agents) so that the software can differentiatedata obtained from the fluorescing sample. As a result, it may bebeneficial to include a filter such as a bandpass filter. Alternatively,a prism device or optical diffusion grating may be utilized forprismatic separation of the light (which may require detectors that willdetect the difference between the light from one or more fluorescingagents and the light from the light source so that data from each can beseparated).

As mentioned above, one possible approach is to employ a plural bandbandpass filter. A plural band (in other words, multi-band) bandpassfilter may be employed in conjunction with either or both the lightsource or detector. The band amount can be selected to correspondgenerally with the number of light sources of different wavelengths usedfor excitation of a sample, or the number of different detectionwavelengths desired. For example, the employment of an excitationquad-band bandpass filter (if a four light source is employed) may beadvantageous. Alternatively, dual-band or tri-band bandpass filters maybe partially employed to minimize the number of com_(p)onentsas comparedto a design using single band bandpass filters. Such a multi-bandbandpass filter may suitably be employed. Such a filter may be sized tobe within a predetermined size (e.g., covering an area that is only aportion of the total area of the array that defines the detector). Forexample, a detector may include an array of a predetermined number ofpixels adapted for detection. However, the filter may be sized forallowing transmission of light to only a fraction of the pixels (e.g.,less than about 75%, less than about 50%, less than about 25%, less thanabout 10% or even less than about 5% of the pixels) available fordetection.

As also discussed above, the light source selected may include groupingsof two or more, preferably at least four, LEDs for ease of providingequal light into a fiber optic cable and ease of printed circuit boarddesign. In addition, one or more light source bandpass filters may beutilized. Preferably, multi-band bandpass filters, more preferably quadband bandpass filters, may be utilized. The multi-band bandpass filtersmay be hard-coated for maximal resistance to heat and humidity. Themulti-band bandpass filters may be adapted to allow light in certainwavelength regions to reach the PCR samples for fluorescent dye/probeexcitation but block wavelength regions where those fluorescentdyes/probes emit light to maximize the signal-to-noise ratio in thedetection signal. For example, the multi-band bandpasss filters may be aquad-band bandpass filter which is hard-coated to allow light with atleast 80% transmission (or even greater than 95% transmission) in the460 nm to 500 nm region, the 510 nm to 535 nm region, the 570 nm to 590nm region, and the 640 nm to 690 nm region while allowing less than 1%transmission, less than 0.1% transmission, or even less than 0.01%transmission in the rest of the visible wavelength spectrum. The opticaldensity of that filter in the transmission regions could be 0.25, 0.1,0.05, 0.02, 0.01 or lower. The optical density between the transmissionregions could 3, 4, 5, or even 6 or higher. A second hard-coatedquad-band bandpass filter may allow light with at least 80% transmission(or even greater than 95% transmission) in the 405 nm to 460 nm region,the 485 nm to 510 nm region, the 535 nm to 565 nm region, and the 610 nmto 650 nm region while allowing less than 1% transmission, less than0.1% transmission, or even leas than 0.01% transmission in the rest ofthe visible wavelength spectrum. The optical density of that secondfilter in the transmission regions could be 0.25, 0.1, 0.05, 0.02, 0.01or lower. The optical density between the transmission regions could be3, 4, 5, or even 6 or higher. It will be apparent to those of skill inthe art that slight variations (up to about 5 nm or even 10 nm) inwavelength cutoffs of the multi-bandpass filters would still beacceptable for exciting florescent dyes/probes or that any of thetransmission regions its the above quad-band filters could be swapped inany combination without any harm to the performance of the opticaldetection system. Such quad-band bandpaas filters can be custommanufactured by any number of companies such as Omega Optical, Inc.,Brattleboro, Vt.; Evaporated Coatings, Inc, Willow Grove, Pa.; or Delta,Copenhagen, Denmark.

Preferably, the instrument is capable of providing for at least fourexcitation colors for each sample and is further capable of detecting atleast four emission colors from each sample (see Table 1 for a shortlist of commonly used dyes/probes). As shown in Table 1, onlyapproximate peak wavelengths are listed, but the reporter dyes/probescan be used outside that wavelength range, albeit less efficiently. Themost commonly used fluorescing agents require blue (480 nm to 495 nm),green (520 nm to 550 nm), amber (585 nm to 615 nm), and red (640 nm to660 nm) excitation light. It is expected that the real-time opticaldetection be capable of producing light at or quite near those fourwavelengths. A five or six color instrument would be preferred.

TABLE 1 Table 1: Commonly used fluorescing agents, their excitationwavelength peaks, and their emission wavelength peaks. Generally,real-time PCR will function if the cycler can emit and detect within ±10nm to 20 nm of the peak. Excitation/Absorption Emission/Detection PeakFluorescing Agent Peak (nm) (nm) AMCA 353 (violet) 442 (indigo) Cyan 500450 (indigo) 500 (blue green) Fluorescein 483 to 495 (blue) 520 to 533(green) FAM 483 to 495 (blue) 520 to 533 (green) SYBR Green I/ 480 to500 (blue) 520 to 530 (green) Rhodamine green Tet 521 (green) 536(green) VIC/HEX/JOE 523 to 535 (green) 555 to 568 (green/green- yellow)NED 546 (green) 575 (yellow) Cy3 550 (green-yellow) 570 (yellow) TAMRA553 (green-yellow) 576 (yellow) Red 610 558 (yellow) 610 (orange-red)ROX/Texas Red 585 (yellow-orange) 605 (orange) Red 640 615 (orange-red)640 (red) Cy5 649 (red) 670 (red) Cy7 743 (red) 767 (red) Licor IRDyes651 to 778 668 to 794 (red - infrared) (several) (red - infrared)

As another embodiment, the bandpass filters could be any combination ofsingle, dual-band, or quad-band bandpass filters with at least 80%transmission in these range: 415 nm to 450 nm for excitation of Atto425, Alexa fluor 430, and similar fluorescing agents with acorresponding detection bandpass filter with at least 80% transmissionthe 470 nm to 510 nm range for the appropriate detection of thosefluorescing agents; 460 nm to 495 nm for excitation of FAM, SYBR GreenI, and similar fluorescing agents with a corresponding detectionbandpass filter with at least 80% transmission in the 515 nm to 548 nmrange for the appropriate detection of those fluorescing agents; 480 nmto 505 nm for excitation of SYBR Green I, Rhodamine green Oregon Green514, and similar fluorescing agents with a corresponding detectionbandpass filter with at least 80% transmission in the 525 nm to 565 nmrange for the appropriate detection of those fluorescing agents: 505 nmto 533 nm for excitation of JOE, VIC, HEX, and similar fluorescingagents with a corresponding detection bandpass filter with at least 80%transmission in the 553 nm to 600 nm range for the appropriate detectionof those fluorescing agents; 530 nm to 555 nm for excitation of NED,TAMRA, Cy3, Rhodamine Red, and similar fluorescing agents with acorresponding detection bandpass filter with at least 80% transmissionin the 575 nm to 620 nm range for the appropriate detection of thosefluorescing agents; 568 nm to 589 nm for excitation of ROX, Texas Red,Red 610, and similar fluorescing agents with a corresponding detectionbandpass filter with at least 80% transmission in the 600 nm to 660 nmrange for the appropriate detection of those fluorescing agents; 605 nmto 645 nm for excitation of Cy5 and similar fluorescing agents with acorresponding detection bandpass filter with at least 80% transmissionin the 665 nm to 705 nm range for the appropriate detection of thosefluorescing agents; and 650 nm to 680 for excitation of Cy5.5, Quasar705, and similar fluorescing agents with a corresponding detectionbandpass filter with at least 80% transmission in the 700 nm to 780 nmrange for the appropriate detection of those fluorescing agents.

Such detection filter may be sized to be within a predermined size(e.g., covering an area that is only a portion of the total area of thephotodiode detector device that defines the detector). For example, adetector device may include an array of a predetermined number of pixelsor individual photodiodes adapted for detection. However, the filter maybe sized for allowing transmission of light to only a fraction of thepixels (e.g., leas than about 75%, less than about 50%, less than about25%, less than about 10% or even less than about 5%) available fordetection.

Another consideration that may prove beneficial in the performance andoperation of the systems herein is the location, angular disposition,and/or spacing of any filter relative to any light source, the sample(and/or holder), the detector, or any combination thereof. For example,suppose that a filter includes generally planar opposing surfaces,though substantial perpendicularity may be possible. There may be anangular disposition of the filter that is less than or greater thanperpendicular by at least about 5, 10, 20, 30, 45° or more relative tothe principal emission axis from the light source.

One or more filters employed herein may be such that they transmit atleast a portion of the emitted visible light (e.g., at least about 50%,65%, 75% or even 90%), as measured using a suitable spectrometer foroptical metrology transmission measurement. One or more filters may beselected to absorb all or substantially all visible light.

It may be desirable for such filters, or one or more other noisereduction filters used in combination therewith to be selected so thatwavelengths that translate as background noise are reduced as comparedwith a system that omits any such filter. By way of example, it is oneobject to substantially reduce or even eliminate any significantcontribution to background noise that may result from infrared radiationarising from any light source used. Desirably, one or more filters maybe employed to substantially block infrared radiation (e.g., to blocktransmission of infrared radiation to a level that is less than about50%, 60%, 70%, 80%, or even of the total infrared radiation that seeksto be transmitted). Thus, one possible approach may be to employ afilter that transmits at least about 70%, 80% or 90% visible light, butabsorbs or otherwise blocks infrared radiation to a level that is lessthan about 30%, 20% or even 10%.

Among the various filter types that may be employed herein are thosesuch as hot mirrors, heat absorbing glass, shortpass filters, longpassfilters, infrared cutoff filters, and wide bandwidth bandpass filters.

Filters herein may have a first face, a generally opposing (e.g.,generally parallel) second face, and a periphery that typically spansbetween the first and second face. It is possible that one or more ofany of the filters herein may be at least partially encapsulated (e.g.,about at least a portion of its periphry) by a material that differsfrom the filter. One or more of any of the filters may include asuitable filter alignment holder. Such holder may be adapted to attachto one or more of the other components of the system. For instance, theholder may be sized and configured to receive one or more filters, andmay also include an attachment portion (e.g., as part of and/oradjoining a peripheral portion of the holder) that includes suitablestructure for attaching the holder within the system. For example, theholder may be such that it can be positioned between a light source anda sample holder, between a sample holder and a detector, or both. Adepiction of one example of a filter alignment holder is shown in FIG.14. The material of the filter alignment holder may be suitable forwithstanding the temperature fluctuations to which it may be subjectedduring operation, such as for avoiding thermal distortion to it, anyfilter it holds, or both. One approach is to employ a material that hasa generally anti-reflective outer surface for avoiding stray emissions.For example, the outer surface to which any light may be directed may beblack. Other components within the system (e.g., the one or more boardsupon which the circuits are printed) may also be coated or selected tobe relatively light absorptive (e.g., they may be generally opaque, suchas a black material).

The filter alignment holder may be coupled with the light source. Forexample, the light source may be supported upon, and/or integrated withone or more circuit boards. One or more filter alignment holder may becoupled with such one or more of such circuit boards. Such coupling maybe a permanent coupling that would effectively require destruction ofone of the components for detachment. Such coupling may be temporary orremovable. For example, one approach may be to employ one or more of anadhesive, a fastener (e.g., a mechanical fastener such as a push pinfastener, a threaded nut assembly, a pin, a clamp or otherwise), a weld(e.g., a stake weld) or any combination of the above. For example, oneapproach may be to employ a gasket, a layer of adhesive that has acertain amount of compliance and will withstand compression and thermalcycling without distortion, or both. Any filter alignment holder, orcombination of filter holders may also have a thickness dimension thatis selected to adjust the angle of excitation light that enters thefiber optics and the amount of emission light that reaches thedetectors. An example of a suitable material for a filter alignmentholder includes thermoplastic or thermoset polymeric containingmaterials. They may be filled and/or reinforced (e.g., with glassfibers) to help foster dimensional stability during cycling. The filteralignment holder may be made of a material with high thermal mass suchthat it bel maintain the temperature of the LEDs and detectors at aconstant temperature. It may also be desirable to help isolate anymetallic materials of the holders from any adjoining circuit boards tohelp reduce the possibility of electrical interference or shortcircuits. The filter alignment holder may be assembled in a manner thatit includes respective filters for performing the respective functionsof filtering light transmitted to a sample, and filtering light emittedby the sample. The filter alignment holder may also include one or moreother filter portions that filter background light.

One approach is to employ a filter alignment holder that includes adifferent thickness for a transmission portion that transmits light tothe sample as compared with the thickness of the holder sample emissionportion through which light is emitted to the detector. The transmissionportion, for instance desirably will be thick enough to allow light toenter at a relatively small angle of incidence from the light source(e.g., below the angle of incidence of a bandpass filter, which is about5°). The holder sample emission portion desirably will be relativelythin (e.g., less than about 90%, 75% or even 20% of the thickness of thesample emission portion). The width of the incident area of lightemitted from an emission optical fiber is a function of the distancebetween the terminal end of the fiber and the detector. Since thedetector elements are fixed in size (such as about 55 μm by 65 μm), thefurther the terminal end of optical fiber is located from the detector,the less light that will reach the detector. The optimal thickness ofthe holder sample emission portion is thus determined by the width ofthe emission optical fiber bundle and the width of the detectorelements. The optimal thickness is also limited by the thickness of thedetection filter (such as a linear variable filter or a set of bandpassfilters) which may be 1 mm, 2 mm, or even 3 mm thick. One preferreddesign may have the detection filter directly deposited onto theterminal ends of the optical fibers and thus the filter thickness isnegligible. Alternatively, the light source circuitry and the lightdetection circuitry could be placed on separate circuit boards and therelevant transmission and emission filter holder sections could beoptically isolated from each other as a single unit or as separatefilter alignment holder components. In this manner, it is believedpossible to help reduce the amount of light from the light source thatreaches any detector without first exciting a sample to achieve sampleemission.

The ininstrument may include lenses that are present within the opticalcomponentry. More specifically, a lens may be present between thevarious optical components to improve the efficiency of the excitationand emission means. A lens or lens assembly may be present between thelight emitting diode and the excitation bandpass filter, such that thespreading light is converged into substantially parallel beams passingthrough the filter. An additional converging len (e.g., biconvex) may bepresent between the excitation filter and the waveguide to focus theseresulting beams into the waveguide. Such lenses allow for more efficientuse of the LED light and homogenize the light entering into the fiberoptic waveguide for improved sample excitation. Further, a similar lensarrangement may be employed on the detector side. Light exiting theemission fork portion that is becoming diffuse due to angular incidencemay be focused and optimally directed through a detection bandpassfilter by a converging lens present there-between. Another converginglens may be present between the detection bandpass filter and photodiodeto further focus the light passing through the detection bandpass filteronto the photodiode so that a high efficiency of signal detection isachieved. A lens may also be present between the sample tube and theterminal end of the waveguide proximate thereto. A lens may beintegrated into any of the other components, such as attached to thebandpass filter. The instrument may be void of a lens between any of theoptical components.

It may also be desirable in the invention to include means foraddressing background noise phenomena. A certain level of backgroundnoise is commensurate with detector reading and leads to a baselinesignal offset which may typically be subtracted during data processing.Due to the temperature dependency and other factors associated with theoptical components, signal noise and baseline drift may be experiencedduring the execution of a real-time protocol. In this sense, it isadvantageous to include means for accounting for baseline noise anddrift. The use of a reference dye is one such aspect for normalizingsignal reads at each cycle. Notwithstanding, it is also contemplatedthat a separate detector or simply some pixels of each photo-diode arrayserve as a reference from which any baseline drift may be furthercorrected.

The teachings herein include componentry enabling for high-speed realtime polymerase chain reaction in a mobile, relatively compactinstrument. The instrument includes one or more components adapted fortransmitting light emitted from a light source (e.g., a light emittingdiode device) to one or more samples located in a sample holder. Theinstrument includes one or more components adapted for transmittinglight emitted from at least one fluorescing agent to one or moredetectors. A filter (e.g., a linear variable filter) may filter anylight emitted by fluorescing material within the one or more samplesprior to reaching the detector. A filter (e.g. quad band-pass filter)may filter the light emitted from the light source prior to reaching thesample.

As shown for example in FIGS. 1A and 1B, the instrument may include aninstrument component portion 10 for housing components including fiberoptic and electric componentry 16. The fiber optic and electriccomponents may be located within the instrument below the thermalcycling assembly 12. As shown in FIG. 1A, one or more panels (which mayform a sheath) 14 may be included for enclosing the components and forkeeping proper airflow to maintain proper component temperature. FIG. 1Bshows the components within the instrument with the panels 14 enclosingthe components made transparent to expose the fiber optic waveguide andelectric componentry 16. One or more electrical components are mountedto a printed circuit board 18.

FIG. 2 shows an exploded view of the instrument component portion 10. Aplurality of panels 14 is shown for enclosing components therein. Thethermocycling assembly 12 is shown above a fiber optics module 20, whichis shown above the electronics 24, which together comprise the fiberoptic and electrical components 16. The electronics are shown mounted toa platform 18, which may include a printed circuit board (PCB). A fan 22that is generally located at the back of the instrument componentportion 10 is also shown.

FIG. 3 shows an exploded view illustration of the internal fiber opticand electrical components of the fiber optics module 20. A sample holder26 is shown for locating within the thermocycling assembly 12 (notshown). A resistance temperature detector (RTD) guide 28 is also shownwhich travels through a fiber optics block 34 (see FIG. 15 for anadditional view of the fiber optics block). A fiber optic cap 30 may belocated over and/or onto the fiber optic block 34. A waveguide includingone or more fiber optics 32 are located within the fiber optics block34. The fiber optics 32 may be formed as bifurcated fiber optics, suchthat a first fork 32 a locates over a light source, and a second fork 32b locates over a detector. The first and second fork converge with oneanother to form a joined arm 32 c which is rotated about 90° from thedirection of the first and second arm and extends upward toward one ormore samples. A bottom cover 36 is located in contact with a bottom edgeof the fiber optic block 34. One or more detection filters 38 may belocated below the bottom cover and aligned with one or more photodiodearrays 44. One or more light source bandpass filters 40 may be locatedbelow the bottom cover and aligned with one or more light sources 46.The one or more detection filters 38 and one or more light sourcebandpass filters 40 may be located onto a filter alignment holder 42(see FIG. 14 for an additional view of the filter alignment holder). Thefilter alignment holder 42 may be located in contact with an optics PCB48, which may include one or more detectors 44 that correspond to theone or more detection filters 38 and one or more light sources 46 thatcorrespond to or more light source bandpass filters 40.

FIGS. 4A and 4B depict an example fiber optics block 34, shown from atop-down view of the fiber optics block. FIG. 4A shows the fiber opticsblock prior to locating the fiber optics cap onto the block, while FIG.4B shows the fiber optics cap 30 located onto the block. The fiberoptics block is shown including a plurality of recesses 50 for receivingthe fiber optics 32 (not shown) and then receiving the fiber optics cap30 (as shown in detail at FIG. 6). FIGS. 5A and 5B show the fiber opticsblock 34 as viewed from beneath the block. The RTD guide 28 is shownextending into the bottom of the block. FIG. 5A shows the block prior toaddition of the bottom cover, whereas the bottom cover 36 is shownlocated onto the block in FIG. 5B. The bottom cover 36 (as shown indetail in FIG. 7) may include an opening 52 for receiving and/or beingsubstantially aligned with the RTD guide (as shown in detail in FIG.10).

FIG. 8 shows an additional top-down view of an example fiber opticsblock 34, while FIG. 9 shows an additional bottom-up view of the fiberoptics block. The views include the plurality of recesses 50 foraligning with and receiving the fiber optics cap 30. The resulting gapformed between the block 34 and the cap 30 forms an opening for thefiber optics 32 to align directly under each sample in the sample holder26. One or more gaps may be formed between the block 34 and the cap 30.Each gap is an opening for the fiber optics 32 to align directly undereach sample in the sample holder 26. An opening 56 may be formed in theblock for receiving the RTD guide. The opening 56 may be located in asubstantially centralized location along the block 34. The block mayfurther include a plurality of openings 54 adapted for attaching theblock to a surface within the instrument. The block 34 may be pottedwith a resin or similar material to permanently keep the assembled blocktogether, to protect the fiber optics 32, and to keep the fiber opticsstationary.

The RTD guide 28 is shown in further detail at FIG. 10. The RTD guidepreferably includes one or more tapered ends 58 for entering into one ormore openings within the fiber optics block. The RTD guide is hollow toallow an RTD (not shown) to pass through the block 34 and measure thetemperature of the sample holder 26.

As discussed with reference to FIG. 3, a printed circuit board (PCB) 48is included within the instrument, an example of which is shown at FIG.11. The PCB includes one or more detectors 44, the number of detectorstimes the number of sample regions per detector corresponding with thenumber of PCR samples per module. For example, the four detectors 44shown each have two sample regions per detector corresponding to eightsamples. The PCB further includes one more light sources 46. In theillustrative embodiment, each light source 46 contains four differentwavelength LEDs. For illustrative purposes, the PCB is shown having twosurfaces 60 to which light sources have not been attached.

In the illustrative figures, each light emitting diode device provideslight to two samples and each detector has two sample regions (i.e. onefor each sample). Other numerical strategies are contemplated. Forexample, each light emitting diode device may provide light to even moresamples, such as four or even eight samples. On the other hand, eachsample could have its own light emitting diode device. In this instanceit would be possible for a detector with only one sample region to beshared by multiple samples by sequential timing of the light emittingdiode devices. Given the short read times required for each sample, asequential sample read strategy may have insignificant impact on totalread time. In the extreme, each sample may have dedicated light sourcesand detectors. However, communal strategies would reduce the totalnumber of components required and thus the required space and overallcost of the instrument.

In one embodiment, for each sample there is one light emitting diodedevice and one photodiode detector device. In another embodiment, forevery two samples there is exactly one light emitting diode device andtwo photodiode detector devices. Alternatively, for every two samplesthere are exactly two light emitting diode devices and one photodiodedetector device. In yet another embodiment, for every four samples thereis one light emitting diode device and four photodiode detector devices.Alternatively, for every four samples there are four light emittingdiode devices and one photodiode detector device. In yet anotherembodiment, for every eight samples there is one light emitting diodedevice and eight photodiode detector devices. Alternatively, for everyeight samples there are eight light emitting diode devices and onephotodiode detector device. In the instances where an unbalanced numberof light emitting diode devices and photodiode detector devicesemployed, the optical reading process may be executed in such a way thatthe signal from each sample can be isolated. By way of illustration, fora shared photodiode detector device the light emitting diode devices areenergized at different times (sample 1 measured, then sample 2, etc.).in a preferred embodiment, there are individual photodiode detectordevices for each sample with shared light emitting diode devices suchthat multiple samples may be read simultaneously.

Another embodiment of the invention may include a step of locating thelight emitting diode device above the sample holder. Excitation of thesample may be provided through transmission of light through the cap andmay include the multiple band pass filter and additional fiber opticcables to transmit the light to the sample. In this instance, the lighttransmission assembly below the sample holder would be comprised ofwaveguides for transmission of light emitted by the fluorescing agent tothe detector.

A flow-chart depicting connectivity of the instrument described hereinwith an illustrative four thermocycling modules shown at FIG. 12. Thesystem architecture diagram 62 shows the central computer 66 ascontrolling the user interface 64. PCR protocols 68 (includingtemperature and optics settings), PCR data 70 (including temperature andoptics data), and the communications board 74. The PCR data 70 is alsoin communication with data analysis software 72 which provide for usabledata storage and statistical analysis resulting from the PCR protocols.The central computer 66 further optionally receives power from a DCpower supply 76 if the computer is internal to the instrument, otherwisethe computer has its own DC power supply. DC power supply 76 receivespower from a filtered AC power source 78. The communications board 74 isin direct two-way communication with each PCR module (depicted in thisexample as 80 a, 80 b, 80 c, and 80 d). Each PCR module also receivespower from the DC power supply 76. Each PCR module providescommunications to a thermoelectric cooler (TEC) device 82 a, 82 b, 82 c,82 d; a light source 84 a, 84 b, 84 c, 84 d; and a fan device 86 a, 86b, 86 c, 86 d. Each PCR module receives communication from one or moretemperature sensors 90 a, 90 b, 90 c, 90 d. Each PCR module is intwo-way communication with one or more light detectors 88 a, 88 b, 88 c,88 d.

Each module may be mostly self-contained. Each module may beindependently controllable and may perform real-time PCR on up to 2, upto 8, up to 12, or even up to 20 samples. The module includes allnecessary electronics and optics (controller board, H-bridge, sensors,thermal protection, LEDs, optical detection hardware, etc) with theexception of the power supply and user interface. Each module may becontained in a sheath (e.g., plurality of panels) 14 for optimum airflowto keep the samples uniform in temperature. The airflow may also go pastall sensitive electronics, detectors, and light sources, keeping themcool. The sheath 14 may also serve as a protective barrier separatingthe hot components, electrified circuits, and static sensitivecomponents from the user and external elements. The module may have one2-wire connector for power, and one 2-wire connector for communicationwith the user-interface electronics. Alternatively the module may haveone 4-wire connector which handles both power and communication. Thisminimal wiring keeps the modules easy to install, maintain, replace,calibrate, and allows for the modules to be easily placed within aninstrument box or as external add-ons to existing equipment. The wiringconnections 94 may extend out the side of the module as shown in FIG.1A, or may extend out of the bottom of the module. The wiringconnections may be plugs and receptacles or pins and matching pinterminals for easy module installation and removal. The modules may befully independent, individually calibrated, may be swapped for easyrepair/maintenance, may be produced in an instrument with 1, 2, 3, 4, ormore modules, and are generally small and portable. The modules may bepre-programmed so that no user interface software or computer isrequired (ideal for medical applications and ease of use). The modulesmay be programmed by a barcode scanner, 2D barcode scanner, NFC (nearfield communications), or RFID (radio frequency identification) from anappropriate test kit, assay, or sample tube.

FIG. 13 shows an example instrument including four separate PCR modules92, each module including a sample holder and each sample holder havingits own fiber optic componentry. FIG. 14 shows an exemplary filteralignment holder 42 showing additional detail for shape and arrangementof openings within the filter holder for receiving filter components.FIG. 15 shows an additional perspective of the fiber optics block 34.FIG. 16 shows a sample block 12 and connected fiber optic and electricalcomponents 16 below.

FIG. 18 shows a side view of an embodiment of the first excitation forkportion. A light emitting diode device 110 shines light through a LEDlens array 108. The LED lens array 108 focuses the light emitted fromthe individual elements of the light emitting diode device 110 throughbandpass filters located in the excitation filter holder 106. Thefiltered light continues to the excitation fiber lens 104 which focusesall of the filtered light beams to the fiber optic bundle 102. The fiberoptic bundle 102 contains at least one optical fiber 100 in a compactstaggered arrangement to minimize the amount of light lost to unusablespace between the optical fibers 100. The optical fiber 100 transmitsthe filtered light to at least one sample tube (not shown) and may beflexible or bent into a permanent configuration to allow for precisealignment with the sample tube. The light emitting diode device 110 maybe integrated assembly of light emitting diodes or the light emittingdiode device 110 may be a compact group of individual light emittingdiodes located on a substrate such as a printed circuit board, with anarrangement such that the light emitting diodes require an area which isless than 3 cm per side, or less than 1 cm per side, or even less thanabout 4 mm per side. The group of light emitting diode elements may haveat least one color, at least 4 colors, at least 5 colors, or at 7colors. The light emitting diode elements may be repeated such that morethan one element has the same color in the light emitting diode device110 to increase intensity of that color and/or to increase the overalllife-span of using that color. The LED lens array 108 could be a singleintegrated component as shown in FIG. 18, or a compact arrangement ofindividual lenses. The lens arrangement in the LED lens array 108 wouldmatch the pattern of light emitting diodes in the light emitting diodedevice.

In one embodiment shown in FIG. 18, the lenses are staggered in the LEDlens array 108. In another embodiment the LED lens array is in arectangular grid arrangement (as example of which is available fromEdmund Optics, Barrington, N.J. under the designation #64-486). Theexcitation filter holder 106 may be a stationary device that holdsindividual bandpass filters. There may be one bandpass filter for eachLED element in the light emitting diode device 110 as shown in FIG. 19.In another embodiment, one or more multi-band bandpass filter may filterlight from more than one LED element in the light emitting diode device110. The excitation filter holder 106 may be a moving filter wheel thatholds bandpass or multi-band bandpass filters as shown in FIG. 20. Theexcitation fiber lens 104 may be a single converging lens designed totransmit light from all of the LED elements to the fiber optic bundle102. In another embodiment the excitation fiber lens may be an array ofindividual lenses. The optical fibers 100 may be designed with arelatively large numercial aperture (0.55 or greater) to maximize thelight that is transmitted to the samples. The optical fibers 100 may beplastic or they may be glass such as borosilicate selected to have highlight transmission in the about 400 nm to about 700 nm range. Theoptical fibers 100 may have a diameter of about 0.1 mm, 0.25 mm, 0.5 mm,or even 1.0 mm. The optical fibers 100 may all go to a single sampletube or the optical fibers may be split to go to more than one sampletube. The optical fibers may go to the top of the sample tube so thatthe light can be transmitted through the optically clear cap of thesample tube. The optical fibers may go to the bottom of the sample tubeso that the light can be transmitted through the optically clear bottomportion of the sample tube. The grouping of the light emitting diodedevice 110, LED lens array 108, excitation filter holder 106, excitationfiber lens 104, and optionally one end of the fiber optic bundle 102 maybe enclosed in a filter alignment holder (not shown). The filteralignment holder blocks light from entering or escaping the firstexcitation fork portion other than through the optic fibers 100; holdsthe excitation fiber lens 104, filter holder 106, and LED lens array 108in proper alignment with the light emitting diode device 110 and thefiber optic bundle 102; and can withstand and potentially dissipate heatgenerated from the light emitting diode device 110.

FIG. 19 shows a perspective view of one preferred embodiment of thefirst excitation fork portion. There are seven light emitting diodeslocated in a staggered arrangement on the light emitting diode device110. Each light emitting diode element is approximately 1 mm×1 mm suchthat the entire hexagonal grouping of light emitting diode elementsoccupies a space of less than about 4 mm×4 mm. The LED lens array 108 isa single optically clear array which is staggered such that each lens isapproximately centered over the top of the individual light emittingdiode elements of the light emitting diode device 110. The excitationfilter holder 106 contains seven bandpass filters with an opticaldensity of less than 1, less than 0.1, or even less than 0.01 in thedesired wavelength ranges and an optical density of greater than 4,greater than 5, or even greater than 6 in the undesired wavelengthranges. There are up to seven different bandpass filters to match the upto seven different light emitting diode elements. A single excitationfiber lens 104 focuses the seven filtered light paths to a grouping of32 staggered optical fibers 100 of 0.33 mm diameter and a 0.55 numericalaperture which are tightly grouped in the fiber optic bundle 102. Thegrouping of 32 staggered optical fibers may be split to transmit lightthrough 32 optical fibers to one sample, 16 optical fibers each to twosamples, eight optical fibers each to four samples, or four opticalfibers each to eight samples. Another embodiment may use 16 opticalfibers with each sample receiving light from 16, 8, 4, or 2 opticalfibers respectively depending on the number of samples. Anotherembodiment may use 64 optical fibers with each sample receiving lightfrom 64, 32, 16, or 8 optical fibers respectively.

FIG. 20 shows a side view and perspective view of another preferredembodiment of the first excitation fork portion. There are seven lightemitting diodes located in a staggered arrangement on the light emittingdiode device 110. Each light emitting diode element is approximately 1mm ×1 mm such that the entire hexagonal grouping of light emitting diodeelements occupies a space of less than 4 mm×4 mm. The LED lens array 108is a single optically clear array which is staggered such that each lensis approximately centered over the top of the individual light emittingdiode elements of the light emitting diode device 110. The excitationfilter holder 106 is comprised of an excitation filter wheel 112 whichcontains approximately seven bandpass filters 116 with an opticaldensity of less than 1, less than 0.1, or even less than 0.01 in thedesired wavelength ranges and an optical density of greater than 4,greater than 5, or even greater than 6 in the undesired wavelengthranges and a motor 114 which turns the filter wheel. There are sevendifferent bandpass filters 116 to match the different light emittingdiode elements. A single excitation fiber lens 104 focuses the sevenfiltered light paths to a grouping of optical fibers 100 in the fiberoptic bundle 102. In another embodiment, there may be fewer than sevenbandpass filters if one or more of the light emitting diode element isrepeated to emit approximately the same color in the light emittingdiode device 110. In another embodiment, there may be more than sevenbandpass filters if it is desirable to use more than one waveband fromone or more of the light emitting diode elements to fine-tune thefiltered light.

FIG. 21 is a representative drawing of the light path through theembodiment of FIG. 18 and FIG. 19. Light from the emitting diode 110spreads away from the light emitting diode element and through the LEDto lens space 118. The LED to lens space 118 is small (less than 3 mm,less than 2 mm, or even less than 1 mm) such that the bulk of the lightfrom a single light emitting diode element goes to a single desired lensin the LED lens array 108. Light focused through the LED lens array 108passes through the lens array to excitation filter holder space 120.Light may focus in a crossing pattern through the excitation filterholder 106 as shown in FIG. 21 or the light may be approximatelycollinear and perpendicular to the light emitting diode device 110. Thefiltered light passes through the excitation filter to excitation fiberlens space 122 and then is focused by the excitation fiber lens 104 topass through the excitation fiber lens to fiber optic bundle space 124.The distances of the spaces 118, 120, 122, and 124 are chosen tomaximize the amount of light that reaches the optical fibers 100 at alight angle that the optical fibers can accept depending on thenumerical aperture of the optical fibers and the specific lens designs.The total distance from the light emitting diode device 110 to the fiberoptic bundle 102 may be less than 1 cm, less than 5 cm, or even lessthan 10 cm.

FIG. 22 shows an embodiment of the second detection fork portion.Detection optical fibers 126 collect light from the sample tubes andtransmit that light to the detection fiber to filter lens 128. In FIG.22, one representative light path 142 is shown. The detection fiber tofilter lens 128 could be an array of individual lenses or an integratedlens array. The detection fiber to filter lens 128 focuses the lightfrom the detection optical fibers 126 into an approximately collinearpath that is approximately perpendicular to the detection filter holder130 and the photodiode detector device 134. Detection filter holder 130is comprised of a motor 138 that rotates the filter wheel 136 andvarious detection bandpass filters 140. Light is filtered by thedetection band as filters 140, is focused by filter to detector lens132, and is detected by photodiode detector device 134. The componentsare arranged in FIG. 22 such that each detection fiber 126 has onedetection to filter lens 128, one detection bandpass filter 140, onefilter to detector lens 132, and one photodiode detector 34. However,any other configurations also may be utilized. More than one detectionfiber 126 could share an optical path through the lenses, filters, andto the detector. A bandpass filter 140 could be large enough to beshared by multiple light paths to reduce the number of componentsrequired. The detection fiber to filter lens 128 and/or filter todetector lens 132 could be individual lenses or an integrated array oflenses. The photodiode detector device 134 could be an integrated devicewith an array of photodiode elements or an array of separate photodiodedetectors. The bandpass filters 140 could be a single wavelength band orcould be multi-band bandpass filters to minimize the number ofcomponents and reduce the number of filter wheel rotations that arenecessary. The number of bandpass or multi-band bandpass filters 140determines the number of colors that could be detected which may allowfor 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different colors to bedetected. The bandpass filters could be arranged such that allphotodiode detectors in the photodiode detector device 134 receive thesame light wavelength simultaneously, or they could be arranged suchthat each photodiode in the photodiode detector device receivesdifferent wavelengths of light.

One or both of the detection fiber to filter lens 128 and/or filter todetector lens 132 could be removed if the detection area of thephotodiode detection device 134 is large enough to accept most or all ofthe light spread from the detection optical fibers 126. Minimizing thedistance between the detection optical fibers 128 and the photodiodedetection device 134 will assist in minimizing that light spread. Forexample a distance of 10 mm, 5 mm, or 3 mm or less is desirable. In FIG.22 the components (fiber optics, lenses, bandpass filters, andphotodiode detectors) are arranged in two rows of four. However, manyother configurations exist. The shape of the grouping could be onesingle row of eight, a curved row, or other grouping such as a round orsquare ngement. Multiple detection fibers 126 could share the samelenses, filters, and photodiode to increase the light sensitivity. Thedetection filter wheel 136 could be distinct and separate from theexcitation filter wheel 112 or they could be the same filter wheel whichshares the same motor 114/138. This combination of filter wheels 112 and136 minimizes the number of components and ensures simultaneous timingof the excitation and detection filters. The entire second detectionfork portion is enclosed in a detection filter alignment holder (notshown). The detection filter alignment holder blocks light from thesurroundings, especially any light that may escape from the firstexcitation fork portion. All or part of detection filter alignmentholder may act as a thermal insulator such that the photodiode detectiondevice 134 will not be impacted by higher temperatures generated in theinstrument. All of part of the detection filter alignment holder may actas a thermal conductor to remove heat generated by the photodiodedetection device 134.

FIG. 23 shows another embodiment of the second detection fork portion.In this case the filter to detector lens 132 is large enough to beshared by all light paths from all detection fibers 126. The largefilter to detector lens 132 focuses light onto a single photodiodedetection device 134. This arrangement allows for a reduction in thenumber of components and assurance that the photodiode detection devicesensitivity is the same for all light paths. The large filter todetector lens 132 could be a single converging filter as shown in FIG.23 or a lens array of individual lenses which focuses each detectionlight path onto the same photodiode detection device 134.

FIG. 24 shows the bottom of a sample tube. This sample tube hasoptically clear bottom portion which has a low optical density of lessthan 1, less than 0.1, or even less than 0.01 to light in the about 450nm to about 750 nm wavelength range. The tube bottom 150 is comprised ofa flat tube portion 146 and an angled/curved tube portion 148. The lightcan transfer to/from the sample within the tube and to/from opticalfibers underneath the tube through the flat tube portion 146 withminimal light reflection, light distortion, and light absorption due tothe flat, thin, and parallel inner/outer surfaces of the sample tubebottom. Optical fibers 142 carry excitation light from the firstexcitation fork portion of the optical manifold 100, within fiber opticbundle 102, and transmit that light into the sample tube. Two potentialconfigurations of the optical fibers 142 are shown in FIG. 24. In thetop image of FIG. 24, there are four excitation optical fibers 142, twoon either side of the flat tube portion 146; in the bottom image of FIG.24 there are eight excitation optical fibers 142, four on either side ofthe flat tube portion 146. The optical fibers are shown to be of maximaldiameter in each configuration to fit within the flat tube portion 146in order to maximize the amount of light that is transferred perexcitation optical fiber. The excitation optical fibers 142 have adiameter of about 0.33 mm and about 0.25 mm respectively in FIG. 24.There may be other configurations in which there are additional, butsmaller, excitation optical fibers 142 to increase the surface areawhich is covered by the excitation optical fibers 142 and thereforeincrease amount of light that can be transferred. The optical fibersalso extend into the angled/curved tube portion 148; however, there isdiminished light transmission in those regions. A large diameteremission optical fiber 144 is centered underneath the flat tube portion146. The large diameter and placement of the emission optical fiber 144allows for maximum surface area and therefore maximum amount offluorophore emission light to be passed through a single fiber. Thediameter may be about 0.5 mm, about 0.75 mm, about 1.0 mm, oe even about1.25 mm. In another configuration, there may be more than oneemissionoptical fiber 144 with diameters which are smaller in order toremain within or mostly within the flat tube portion 146. For example,there may be four emission optical fibers 144 which are about 0.5 mm indiameter and placed within the center of the flat tube portion 146. Theemission optical fiber 144 may be round as shown in FIG. 24 or it may bea molded optically transparent plastic with a more rectangular shapewhich acts as a light pipe. The emission optical fiber may be opticallytransparent to light with an optical density of less than 1, less than0.1, or even less than 0.01 in the about 450 nm to about 750 nmwavelenth range.

As to all of the foregoing general teachings, as used herein, unlessotherwise stated, the teachings envision that any member of a genus(list) may be excluded from the genus; and/or any member of a Markushgrouping may be excluded from the grouping.

Unless otherwise stated, any numerical values recited herein include allvalues from the lower value to the upper value in increments of one unitprovided that there is a separation of at least 2 units between anylower value and higher value. As an example, if it is stated that theamount of a component, a property, or a value of a process variable suchas, for example, temperature, pressure, time and the like is, forexample, from 1 to 90, preferably from 20 to 80, more preferably from 30to 70, it is intended that intermediate range values such as (forexample, 15 to 85, 22 to 68, 43 to 51, 30 to 32 etc.) are within theteachings of this specification. Likewise, individual intermediatevalues ere also within the present teachings. For values which are lessthan one, one unit is considered to be 0.0001, 0.001, 0.01 or 0.1 asappropriate. These are only examples of what is specifically intendedand all possible combinations of numerical values between the lowestvalue and the highest value enumerated are to be considered to beexpressly stated in this application in a similar manner. As can beseen, the teaching of amounts expressed as “parts by weight” herein alsocontemplates the same ranges expressed in terms of percent by weight.Thus, an expression in the Detailed Description of the Invention of arange in terms of at “‘x’ parts by weight of the resulting polymericblend composition” also contemplates a teaching of ranges of samerecited amount of “x” in percent by weight of the resulting polymericblend composition.”

Unless otherwise stated, all ranges include both endpoints and allnumbers between the endpoints. The use of “about” or “approximately” inconnection with a range applies to both ends of the range. Thus, “about20 to 30” is intended to cover “about 20 to about 30”, inclusive of atleast the specified endpoints. Concentrations of ingredients identifiedin Tables herein may vary ±10%, or even 20% or more and remain withinthe teachings,.

The disclosures of all articles and references, including patentapplications and publications, are incorporated by reference for allpurposes. The term “consisting essentially of” to describe a combinationshall include the elements, ingredients, components or steps identified,and such other elements ingredients, components or steps that do notmaterially affect the basic and novel characteristics of thecombination. The use of the terms “comprising” or “including” todescribe combinations of elements, ingredients, components or stepsherein also contemplates embodiments that consist essentially of, oreven consist of elements, ingredients, components or steps. Pluralelements, ingredients, components or steps can be provided by a singleintegrated element, ingredient, component or step. Alternatively, asingle integrated element, ingredient, component or step might bedivided into separate plural elements, ingredients, components or steps.The disclosure of “a” or “one” to describe an element, ingredient,component or step is not intended to foreclose additional elements,ingredients, components or steps.

It is understood that the above description is intended to beillustrative and not restrictive. Many embodiments as well as manyapplications besides the examples provided will be apparent to those ofskill in the art upon reading the above description. The scope of theinvention should, therefore, be determined not with reference to theabove description, but should instead be determined with reference tothe appended claims, along with the full scope of equivalents to whichsuch claims are entitled. The disclosures of all articles andreferences, including patent applications and publications, areincorporated by reference for all purposes. The omission in thefollowing claims of any aspect of subject matter that is disclosedherein is not a disclaimer of such subject matter, nor should it beregarded that the inventors did not consider such subject matter to bepart of the disclosed inventive subject matter.

What is claimed is:
 1. An instrument for performing polymerase chainreaction with real-time detection comprising: a) a polymerase chainreaction instrument that includes a sample holder having one or moresample wells, wherein the one or more sample wells are configured toreceive one or more sample tubes that each have at least one portionthat is generally optically transparent, and that receives a biologicalsample having a nucleic acid to be amplified and at least onefluorescing agent that interacts with the nucleic acid duringamplification and that emits light upon excitation by light of a knownwavelength; b) at least one light emitting diode device that is carriedon at least one support substrate, is in electrical communication with apower source, is adapted to emit light at a plurality of differentwavelengths, and is located below the sample holder so that the sampleholder receives light emitted from the at least one light emitting diodedevice at a bottom of the sample holder; c) at least one photodiodedetector device located below the sample holder and which is adapted toissue signals based upon intensity of light it receives; d) a lighttransmission assembly that includes at least one waveguide which ismulti-branched and extends towards or partially into the one or moresample wells of the sample holder, wherein the al least one waveguideincludes: i) at least one first excitation fork portion that extendsbetween the sample holder and the at least one light emitting diodedevice for transmitting light emitted from the at least one lightemitting diode device to the biological sample contained in the sampleholder to excite the at least one fluorescing agent; and ii) at leastone second emission fork portion that extends between the sample holderand the at least one photodiode detector device for transmitting lightemitted by the at least one fluorescing agent upon its excitation andhaving a first end that is proximate the sample holder and a second endthat is proximate the at least one photodiode detector device; and e)one or more emission filters disposed between the second end of the atleast one second emission fork portion and the at least one photodiodedetector device and is adapted to filter the light emitted by the atleast one fluorescing agent across a plurality of bandwidths so that thewavelengths of light received by the at least one photodiode detectordevice are known, wherein the one or more filters include a linearvariable band pass filter, series of bandpass filters, or multiple bandbandpass filter. 2-20. (canceled)
 21. The instrument of claim 1, whereinthe instrument includes one or more excitation filters disposed betweenan end of the first excitation fork portion and the at least one lightemitting diode device which are adapted to filter the light emitted bythe at least one light emitting diode device into multiple bandwidthsfor excitation of the biological sample.
 22. The instrument of claim 1,wherein the instrument includes a rotatable filter wheel which includesthe one or more emission filters.
 23. The instrument of claim 21,wherein the instrument includes a rotatable filter wheel which includesthe one or more excitation filters.
 24. The instrument of claim 21,wherein the instrument includes a rotatable filter wheel which includesthe one or more emission filters and the one or more excitation filters.25. The instrument of claim 1, wherein the instrument includes opposingthermoelectric devices which sandwich the sample holder.
 26. Theinstrument of claim 1, wherein the instrument includes a housing betweenthe sample holder and the at least one support substrate which receivesthe at least one waveguide.
 27. The instrument of claim 26, wherein thehousing includes one or more chambers which isolate the at least onephotodiode detector device from the at least one light emitting diodedevice.
 28. The instrument of claim 1, wherein the at least onewaveguide partially extends into the one or more sample wells.
 29. Theinstrument of claim 1, wherein the at least one waveguide includes oneor more fiber optic bundles.
 30. The instrument of claim 1, wherein theat least one waveguide includes a joined armed which extends into atleast one bifurcated portion, and the bifurcated portion includes the atleast one first excitation fork portion and the at least one secondemission fork portion.
 31. The instrument of claim 1, wherein the atleast one light emitting diode device is an integrated assembly of LEDsor a compact group of light emitting diodes.
 32. The instrument of claim1, wherein the at least one light emitting diode device is less than 1cm on each side.
 33. The instrument of claim 1, wherein the at least onephotodiode detector device is a photodiode pixel array or compactgrouping of photodiodes, such that the wavelengths of light receivedacross individual pixels or photodiodes are of known wavelengthscorresponding to one or more different fluorescent agents present withinthe one or more sample tubes.
 34. The instrument of claim 1, wherein theone or more sample tubes are optically transparent along a bottomportion of the one or more sample tubes.
 35. The instrument of claim 1,wherein each of the at least one light emitting diode device includes atleast 4 light emitting diode elements.
 36. The instrument of claim 1, atleast one converging lens is present between the second end of the atleast one second emission fork portion and the at least one photodiodedetector device.
 37. The instrument of claim 1, wherein one or moreemission filters are configured to be optically aligned the at least onephotodiode array detector.
 38. The instrument of claim 26, wherein thehousing includes an upper portion aligned with the sample holder, and abase portion having a cavity therein through which the at least onewaveguide is passed.
 39. The instrument of claim 1, wherein for eachsample there is one or more of the at least one light emitting diodedevice, one or more of the at least one photodiode detector device, orboth.